Figure 1.
Chomatograms of the three identified MC4R mutations.
Chromatograms of MC4R P272L (A), MC4R N74L (B) and MC4R I251L (C).
Figure 2.
Cellular localization of the distinct MC4R mutations.
A) N2A cells were transfected with wt-HA-MC4R-GFP and mutated HA-MC4R-GFP (N74L, I251L and P272L) or incubated at 37°C for 48 h. Receptor at the cell surface was measured by enzyme-linked immunoassay. Data are expressed as percentage of the wt receptor, (n = 3). B) N2A cells were transiently transfected as in A. Cells were transferred at 4°C, incubated with primary rat monoclonal anti-HA antibodies, fixed, and incubated with secondary Cy3-conjugated anti-rat antibodies. C–E) Quantification of the confocal images of the experiment shown in B. The ratio of HA-MC4R-GFP at the cell surface/total receptor in the cell (E) was estimated by dividing the fluorescence intensity of the receptor at the cell surface (Cy3, C) by the fluorescence intensity of the receptor in the entire cell (GFP, D). wt-HA-MC4R-GFP (n = 64) and mutated HA-MC4R-GFP (N74L n = 111, I251L n = 74 and P272L n = 72). Statistical significance, *** = p<0.0001. F) N2A cells were transiently transfected with wt-HA-MC4R-GFP and mutated HA-MC4R-GFP P272L and incubated at 37°C for 48 h. Cells were fixed, incubated with primary rabbit polyclonal anti-calnexin antibodies (Enzo Life Science, Farmingdale, NY) and with secondary Cy3-conjugated anti-rabbit antibodies. G–H) N2A cells were transiently transfected with wt-HA-MC4R-GFP and mutated HA-MC4R-GFP (N74L, I251L or P272L) and incubated at 37°C for 48 h. Cells were stimulated with 100 nm α-MSH for 15 min at 37°C. Intracellular cAMP was measured by using the immunoassay kit by Assay Designs (San Diego, CA), following the manufacturer's instructions. The amount of cAMP generated was expressed as pmols cAMP/mg prot (G), and pmols cAMP/receptor at the cell surface (H), (n = 3). ).
Figure 3.
Functional studies and rescue of the mutation P272L.
A) N2A cells were transiently transfected with wt-HA-MC4R-GFP and mutated HA-MC4R-GFP-P272L and treated with vehicle or 2 mM PBA, 20 µM UBE-41 or both at the time points indicated in the diagram. B) Receptor at the cell surface from cells in A was measured by a spectrophotometric assay. Data are expressed as percentage of the wt without treatment, (n = 3). C) Intracellular cAMP from cells in A was measured by using the immunoassay kit by Assay Designs (San Diego, CA), following the manufacturer's instructions. The amount of cAMP generated by was expressed as pmols cAMP/mg protein, (n = 3). Statistical significance, * = p<0.05,** = p<0.001,*** = p<0.0001, ns = p>0.05.
Figure 4.
Functional studies comparing different MC4R mutations.
A) N2A cells were transiently co-transfected with mutated HA-MC4R-GFP P272L or HA-MC4R-GFP I316S and FLAG-ubiquitin. Immunoprecipitation (IP) from cell lysates was carried out with anti-HA antibodies and analyzed by Western blot (WB) by using the indicated antibodies. B) The graph shows the quantification of the experiment shown in A. The data are expressed as % of ubiquitinated MC4R obtained from the ratio anti-FLAG immunoreactivity/anti-HA immunoreactivity, (n = 3, including that shown in A). C) N2A cells were transiently transfected with mutated HA-MC4R-GFP P272L or HA-MC4R-GFP I316S and treated with vehicle or 20 µM UBE-41 for 16 h. Receptor at the cell surface was measured by a spectrophotometric assay. Data are expressed as percentage of the obesity-linked MC4R I316S variant without treatment, (n = 3). D) N2A cells were transiently transfected and treated as in A. Cells were transferred at 4°C, incubated with primary rat monoclonal anti-HA antibodies, fixed, and incubated with secondary Cy3-conjugated anti-rat antibodies. E) The graph shows the quantification of the experiment shown in D. (P272L n = 22, P272L+UBE-41 n = 13, n = 15 and I316S+I316S+UBE-41 = 10). D) Statistical significance, ** = p<0.001,*** = p<0.0001.